An RNA polymerase preparation from Methylobacterium extorquens AM1 capable of transcribing from a methylotrophic promoter.
RNA polymerase (RNAP) was purified from Methylobacterium extorquens AM1 cells grown on methanol or on succinate. The beta, beta', alpha and omega subunits were approximately the same size as those of Escherichia coli, and the identity of the omega subunit was confirmed by N-terminal sequence analysis. N-terminal sequence analysis suggested that two other polypeptides in the purified RNAP preparation might be sigma factors, a 40 kDa polypeptide that shared identity with sigma 32 homologues, and a 97 kDa polypeptide that shared identity with sigma 70 homologues in other bacteria. The 97 kDa polypeptide did not cross-react with antibody to E. coli sigma 70. The same complement of putative sigma factors was found in RNAP purified from M. extorquens AM1 grown on succinate and those grown on methanol, indicating that no major methanol-inducible sigma factor is present in this strain. Run-off assays showed that the purified RNAP was capable of initiating transcription specifically at the transcriptional start site of a methylotrophic gene, mxaF, which encodes the large subunit of methanol dehydrogenase and is found only in methylotrophic bacteria.
Davagnino
Juan
J
Department of Chemical Engineering, Box 357242, University of Washington, Seattle, WA 98195-1750, USA.
Springer
Amy L
AL
Department of Chemical Engineering, Box 357242, University of Washington, Seattle, WA 98195-1750, USA.
Lidstrom
Mary E
ME
Box 351750 and Department of Microbiology, Box 357242, University of Washington, Seattle, WA 98195-1750, USA.|Department of Chemical Engineering, Box 357242, University of Washington, Seattle, WA 98195-1750, USA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics*; D046911:Macromolecular Substances; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000737:chemistry; D013386:Succinates / Q000378:metabolism; D014158:Transcription, Genetic
D046911:Macromolecular Substances; D010446:Peptide Fragments; D012808:Sigma Factor; D013386:Succinates; D012321:DNA-Directed RNA Polymerases; D000432:Methanol